Gene-specific probes

In a DNA array, gene-specific probes are created and immobilized on a chip (silicon wafer, nylon or glass array substrate). Biological samples are labeled with fluorescent dyes or radioactivity. These labeled samples are then incubated with the probes to allow hybridizations to take place in a high fidelity manner. After incubation, non-hybridized samples are washed away and spot fluorescent or radioactivity signals resulting from hybridization can be detected. 

As part of a study to quantify contributors of stress to hyperglycemia and ketosis in diabetes, normal hepatocytes and adipocytes in tissue culture were treated with cortisol and analyzed by Northern blotting using a gene-specific probe. The results of one experiment are shown below. 

B2. Borresen, A. L., Hovig, E., etal, Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes. Mutat. Res. 202(1), 77-83 (1988). 

The fluorogenic 5 nuclease assay, which utilises the 5 nuclease activity inherent as a secondary function of Taq DNA polymerase to cleave a gene-specific probe, thereby releasing a fluorescent molecule for detection. 

The expression of CBl RNA can be compared by real-time PCR (TaqMan, ABI) using gene-specific probe and primers. Figure 1 shows the reduced expression of CBl gene transcripts in the striatum, midbrain, and hippocampus following acute and chronic treatment of mice with morphine. 

Fig. 1. C57BL/6 mice were acutely treated with 20 mg/kg morphine subcutaneously and separate sub groups treated chronically with either the same dose of morphine daily or saline. Mice were sacrificed 4 h after final injections and the brains quickly dissected to extract RNA for CBir gene expression study. The expression of CBir RNA was compared by real-time PCR using the gene-specific probe and primers. Acute and chronic treatment with 20 mg/kg morphine produced a reduction in the expression of CBir gene transcripts in the striatum (str), midbrain (mid), and hippocampus (hip).

Transcripts from this RNA polymerase subunit operon have been characterized by Northern hybridization. rpoB, rpoCl, and rpoC2 gene-specific probes all hybridize to... 

Figure 4. Dot-blot hybridization and RT-PCR analysis of peg operon in strain 103, (A) Dot-blot hybridization was performed using gene-specific probes generated by PCR, The sizes of the probes were identical to products obtained by RT-PCR analyses (B), (B) RT-PCR analyses of each gene and (C) RT-PCR analyses of each intergenic region were performed.

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